Although significant advances in the treatment of opiate addiction have been made, relapse to opiate use after abstinence continues to impede successful treatment, highlighting the need for efforts to dissect the mechanism of opiate-dependent changes in brain plasticity. Recent studies have attempted to determine the role of structural plasticity in drug-induced behavior with conflicting findings which may result from the complexity of the intracellular signaling mechanisms underlying structural plasticity of dendritic spines caused by drugs of abuse. In this application we propose to develop novel in vivo approaches that will allow us to image the dynamic structural and functional plasticity that is triggered following opiate exposure and that may play a role in the mechanisms underlying reinstatement of drug seeking. To determine the relationship between dendrite structure and the formation and retrieval of drug associated memories, we propose to investigate how morphine exposure in a novel context modifies hippocampal dendritic spine morphology. In vivo imaging is a powerful tool to track rewiring in the hippocampal neural network, at the level of individual spines, throughout the learning, acquisition, expression, extinction and subsequent reinstatement of morphine conditioned place preference (CPP). Ideally one would be able to observe neural networks, in real time, as morphine CPP and reinstatement take place. The advent of virtual reality training paradigms during two-photon imaging makes this combination of behavior and network surveillance possible. In vivo imaging will enable us to follow the dynamics of spine remodeling and allow us to determine whether spine changes are cause or consequence of CPP. In addition, we will be able to visualize whether alterations in dendritic spines persist even following extinction which would indicate that spine remodeling may help store the latent memory driving drug-context associations. Therefore, the goals proposed in this application are: 1) to use in vivo 2-photon imaging in the dorsal hippocampus to follow structural changes in dendritic spines in CA1 neurons during the acquisition and expression of morphine CPP, and following its reinstatement; 2) to implement novel virtual reality spatial navigation protocols that enable us to conduct structural and functional imaging analyses of hippocampal cells in vivo during the formation of morphine-context associations. Overall, in this proposal we will conduct in vivo imaging analyses in awake mice to elucidate the temporal dynamics of hippocampal dendritic spine remodeling and its relationship to the formation of drug-context associations that may play a role in the mechanisms underlying reinstatement of drug seeking. In addition we will implement novel virtual navigation approaches to examine hippocampal circuit dynamics during the formation of morphine-context associations. The imaging methods and behavioral training protocols pioneered in this grant will be widely disseminated to the addiction field to advance the boundaries of current research.